The tyrosine kinase activity of ErbB receptors is stimulated by the binding of agonist ligand, followed by dimerization of the receptor subunits. Phosphorylation of the subunits occurs in trans in the context of the assembled dimer. The ErbB2/ErbB3 heterodimer is the most highly transforming combination of ErbB receptors. This is unexpected as ErbB2 is kinase active but does not bind a ligand while ErbB3 binds a ligand but is kinase-impaired. Recent studies have suggested that in addition to the traditional back-to-back dimers, this pair forms heterotetramers that allow the ErbB2 in one heterodimer to phosphorylate the ErbB2 in the other heterodimer across the tetrameric structure. This inter-dimer phosphorylation is required for the activation of signals mediated by the binding of SH2 and PTB domain-containing proteins to the C-terminal tail of ErbB2. Beyond requirement for the formation of higher order oligomers, other features of the ErbB2/ErbB3 system differ from those described in the current paradigm, indicating the need for a more refined model that incorporates the increasing complexity of signaling by ErbB receptors. Furthermore, while it is clear that the C-terminal tails of ErbB receptors recruit phosphotyrosine binding proteins, the structural and functional specifics of this step in signaling are completely unknown. The goal of the proposed research is to understand how the different oligomeric states of ErbB2 and ErbB3 are formed and how they regulate the output signal, and to provide a molecular understanding of the structure and dynamics of the C-terminal tail as it is phosphorylated and binds proteins to initiate downstream signaling events. To this end, the specific aims of this grant are to: 1) Develop a model that accurately describes and predicts the dynamics among the oligomeric states of ErbB2 and ErbB3 and determine how this dynamic is altered by tumorigenic mutations in these receptors; 2)!Delineate the role of ErbB2/ErbB3 dimers and tetramers in signal transduction; and, 3) Develop a molecular understanding of how the C-terminal tail of ErbB2 mediates downstream signaling. These aims will be addressed using a combination of in vivo, in vitro and in silico analyses. In particular, we will use atomistic simulations of the C-terminal tail of ErbB2 to generate hypotheses regarding how phosphorylation and the binding of SH2 and PTB domain-containing proteins alter the local and global structure of the tail. These in silico predictions will be tested using a novel luciferase fragment complementation technique that allows us to monitor the binding of specific proteins to the C-terminal tails of ErbB receptors. They will also be tested in vitro by mass spec footprinting of the tails phosphorylated in the absence and presence of phosphotyrosine binding proteins. The interaction of SH2 and PTB domain-containing proteins with phosphotyrosine residues is a signaling mechanism used by a host of hormones and growth factors. The mechanistic insight into this process developed in these studies will provide information universally applicable to many different signaling systems. !